Chapter 4: A Quantitative and Conformational Study of the
4.3.4 Model of VanS A autokinase activity
The results from the ion mobility experiment conducted on non-phosphorylated, phosphorylated and ATP-bound forms of VanSA have been used, a long with prior
knowledge, to suggest a mechanism for VanSAautokinase activity. This is depicted
in Figure 4.16 and is similar to that suggested for HK853 by Marina et al.(Marinaet al.2005).
Figure 4.16: Pictorial representation of VanSA autokinase activity. The catalytic ATP-binding domain is represented in purple whilst the dimerisation phosphotransfer domain is represented in blue. a.) The kinase domain is free to move around the phosphotransfer domain, hinging around the linker region between the two domains. b.) Upon ATP-binding, VanSAfavours a more compact conformation. c.) The kinase domain comes into close proximity to the phosphotransfer domain. d.) This allows for transfer of phosphate from the associated ATP molecule to VanSA(H164). e.) Once phosphorylated, VanSAno longer favours a more compact conformation. The kinase domain is free to move. This allows for ADP release.
The CA domain is proposed to be free to rotate around the DHp domain, hinging around the linker region between the two domains. This results in an array of conformational states being exhibited by the protein. Upon ATP binding, the most compact of the multiple conformations observed is favoured i.e. more protein preferentially occupies this state than any other. This more closed conformation likely permits the interaction between the ATP molecule and the site of phosphorylation, so phosphorylation can occur. No significant differences between the conformations occupied between phosphorylated and non-phosphorylated protein are observed and, therefore, after phosphorylation, the most compact conformation is no longer favoured. It is likely that the ATP-binding domain moves out to allow ADP release. In the cell, this would then permit the interaction between the phosphotransfer domain of VanSAand the response regulator, VanRA.
This model is in agreement with others which have been suggested for different HKs (Marina et al. 2005; Bick et al. 2009) but these results give a further indication of protein dynamics. VanSA exists in similar conformational states whether ATP is
associated or not; ATP-binding is not likely to cause a conformational transition. It is proposed that the bound ATP molecule interacts with the DHp domain when they are sufficiently close in a conformational state.
Ion mobility mass spectrometry has been used to provide information relating to the conformational states of the VanSA protein core under non-phosphorylated, ATP-
associated and phosphorylated conditions. MS has been used to confirm that VanSA
is able to autophosphorylate in the presence of ATP and MgCl2. The rate at which
phosphorylation proceeds and the amount of phosphorylation observed has been studied.
This work confirms that ATP association leads to protein phosphorylation and ADP release. Ion mobility experiments provide data enabling conformational changes occurring during VanSAautophosphorylation to be observed. When in complex with
magnesium and ATP, which are required for phosphorylation to occur, VanSA
appeared to favour a more compact conformation. This conformational state is proposed to facilitate autokinase activity by bringing the ATP molecule and the site of phosphorylation into sufficient proximity for phospho-transfer to occur.
For all VanSAtruncates studied, ion mobility experiments illustrated the presence of
multiple protein conformations. They indicate that, in the gas phase, VanSAΔ155
exists in a larger number of stable conformations than VanSAΔ110. This is supported
by ESI-MS spectra obtained for these two species. The charge-state distribution observed in the spectrum for VanSAΔ110 contains fewer maxima than the charge-
state distribution observed in the spectrum for VanSAΔ155. The presence of multiple
conformations, in solution, provides an explanation as to why histidine kinases have proved so difficult to crystallise. Mass spectrometry could be used as a tool to help determine protein constructs which would crystallise in this case. Mass spectrometry has previously been shown to be a valuable tool for the protein crystallographer as it can be used to verify correct protein expression (Chait 1994), elucidate protein domains (Cohen 1996) and analyse protein crystals (Cohen and Chait 2001).
The use of mass spectrometry to study phosphorylation rates is a valuable technique, which offers a simple, reproducible alternative to conventional biophysical methods.
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